To search for genetic sources of allele-specific mRNA translation, we leveraged heterozygous polymorphisms and variants present in the exome of HCT116 colorectal adenocarcinoma-derived cells, computing allelic fractions from both total and polysome-associated RNA from RNA-Seq data. Allelic imbalance in polysomal RNA led us to nominate 52 coding variants associated with allele-specific mRNA translation, of which 16 are non-synonymous. To validate instances of allele-specific translation, a proteomics workflow was developed that combines label-free shotgun analysis, high-pH reversed-phase peptide fractionation, and targeted parallel reaction monitoring using isotope-labeled peptide standards. Using this approach, we provide proof-of-concept validation of the heterozygous G>A, R183H missense single-nucleotide variant rs1554710467 in the eukaryotic initiation factor 4H (EIF4H) gene. The variant is present in two EIF4H alternatively spliced variants, which showed equivalent translation efficiency in HCT116 cells but differ in abundance. The alternative peptide containing H183 was significantly more abundant than the corresponding reference peptide containing R183, consistent with the over-representation of the alternative allele in polysomal RNA in HCT116 cells. A dual-fluorescence ribosome-stalling assay confirmed the enhanced translation potential of the variant allele. The two EIF4H allelic proteins exhibited similar stability and subpolysomal localization. This study demonstrates the feasibility of using allele-specific proteomics at the endogenous protein levels by exploiting heterozygous coding variants. Overall, our approach extends the toolbox available to investigate allele-specific differences in mRNA translation potential, a relatively underexplored layer of gene expression regulation that could reveal interindividual differences in disease-relevant phenotypes.

Abstract To prioritize germline genetic variants affecting mRNA fate at the post-transcriptional and translational levels, we leveraged sucrose-gradient-based isolation of 80S monosomes and polysomes, followed by mRNA retrieval and paired-end sequencing. Total cytoplasmic RNA was also sequenced for comparison. Experiments were performed in the non-transformed cell line RPE-1, cultured under basal conditions or upon p53 activation by Nutlin. Differential gene expression analysis confirmed a canonical p53 response. Heterozygous SNPs and SNVs were identified from the RNA-seq data, and allelic fractions (AF) were calculated for total, monosomal, and polysomal mRNAs. Variants showing reproducible AF differences across fractions beyond experimental variability were defined as tranSNPs. Among nearly 7000 heterozygous variants analyzable in polysomal or total RNA and over 5000 in monosomal mRNA, 1155 displayed a significant imbalance. Reporter assays performed in both RPE-1 and HCT116 cells validated allelic or haplotype effects for 17 selected variants in UTRs and coding regions, confirming differences in 15 cases, with evidence of cell line-specific responses. Proteomic analysis further supported allelic imbalance for selected missense variants. Overall, tranSNPs were identified in a non-transformed cell line at frequencies comparable to those in cancer cells, thereby extending their implications in human physiology. Further, monosome profiling enabled improved detection sensitivity of tranSNPs without positional bias, suggesting that 80S profiling improves detection of allele-specific translational regulation in RPE-1 cells. Graphical Abstract

G-quadruplexes (G4s) are noncanonical, four-stranded nucleic acid structures formed in guanine-rich regions that influence many biological processes, including transcription. We previously developed a yeast-based system to study the transcriptional modulation of p53 family proteins by G4-forming sequences (G4FS) using artificial constructs. Here, motivated by the observation of genome-wide enrichment of G4FS in proximity to p53 and NF-kappa B response elements (REs), we examined how native combinations of G4FS and REs derived from human promoters influence the activity of p53 or NF-kappa B family proteins. To this end, five pairs of isogenic reporter strains were developed, and the propensity of G4FS to adopt G4 structures was confirmed by in vitro assays. Results in yeast showed that the presence of G4FS enhanced the transcriptional output of partial-function p53 mutants, p63, and p73 relative to wild-type p53, suggesting a general boost in the activity of weaker transcription factor proteins. Conversely, the effect of G4FS on transactivation by NF-kappa B proteins was context-dependent and mainly inhibitory. In conclusion, our findings highlight the importance of DNA topology in transcriptional regulation by both p53 and NF-kappa B proteins and demonstrate that the yeast-based system serves as a valuable tool for isolating the contribution of G4FS within a controlled genomic context.

G-quadruplexes (G4s) are noncanonical, four-stranded nucleic acid structures formed in guanine-rich regions that influence many biological processes, including transcription. We previously developed a yeast-based system to study the transcriptional modulation of p53 family proteins by G4-forming sequences (G4FS) using artificial constructs. Here, motivated by the observation of genome-wide enrichment of G4FS in proximity to p53 and NF-κB response elements (REs), we examined how native combinations of G4FS and REs derived from human promoters influence the activity of p53 or NF-κB family proteins. To this end, five pairs of isogenic reporter strains were developed, and the propensity of G4FS to adopt G4 structures was confirmed by in vitro assays. Results in yeast showed that the presence of G4FS enhanced the transcriptional output of partial-function p53 mutants, p63, and p73 relative to wild-type p53, suggesting a general boost in the activity of weaker transcription factor proteins. Conversely, the effect of G4FS on transactivation by NF-κB proteins was context-dependent and mainly inhibitory. In conclusion, our findings highlight the importance of DNA topology in transcriptional regulation by both p53 and NF-κB proteins and demonstrate that the yeast-based system serves as a valuable tool for isolating the contribution of G4FS within a controlled genomic context.

Relatively few studies have examined the link between SNPs and mRNA translation, despite the established importance of translational regulation in shaping cell phenotypes. We developed a pipeline analyzing the allelic imbalance in total and polysome-bound mRNAs from paired RNA-seq data of HCT116 cells and identified 40 candidate tranSNPs , i.e. SNPs associated with allele-specific translation. Among them, the SNP rs1053639 (T/A) on DNA damage-inducible transcript 4 (DDIT4) 3’UTR was identified, with the reference T allele showing a higher polysome association. rs1053639 TT clones generated by genome editing exhibited significantly higher DDIT4 protein levels than AA ones. The difference in DDIT4 proteins was even greater when cells were treated with Thapsigargin or Nutlin, two perturbations that induce DDIT4 transcription. The RNA binding protein RBMX influenced this allele-dependent differences in DDIT4 protein expression, as shown by RNA-EMSA, RIP, and smiFISH assays. RBMX depletion reduced DDIT4 protein in TT clones to the AA levels. Functionally, TT clones more effectively repressed mTORC1 under ER stress, while AA clones outcompeted TT clones in vitro or when injected in zebrafish embryos. The rs1053639 AA genotype, under a recessive model, correlates with poor prognosis in TCGA cancer data. ### Competing Interest Statement The authors have declared no competing interest.

Small nucleolar RNAs are non-coding RNAs typically encoded within the introns of both protein-coding and non-coding genes. Interestingly, a significant fraction of snoRNA sequences is found as retained introns of specific mRNA isoforms expressed from their host gene. In the present study, we aimed to define the representation of small nucleolar RNA retaining transcripts across various human cell types and tissues including cancer. We found that these type of transcripts are widely represented in normal tissues and cancer-derived cell lines, appearing both in their full-length form and, frequently, in a shorter variant. We characterized the shortening position, which occurs at or very close to the retained small nucleolar RNA sequence at the 5' end. Interestingly, for some transcripts this shorter variant represents the only form detected. In addition, some of the small nucleolar RNA retaining transcripts can be localized into the cellular cytoplasmic fraction. Moreover, our findings point out that a variable but consistent proportion of small nucleolar RNA sequences in cells, tissues, and liquid biopsy samples is, in fact, present as small nucleolar RNA retaining transcripts, indicating that these elements should be carefully considered when snoRNA are evaluated as biomarkers. Considering that short reads and gene-based transcriptomic analysis completely overlooked these transcripts, potentially missing critical insights into their involvement in cancer and other diseases, our results strongly indicate that these type of transcripts should be further investigated in different contexts to better understand their biogenesis, sequence features, presence, and role within cells.

Neuroblastoma (NB) is among the most common malignancies in children and represents a therapeutic challenge in pediatric oncology. p53 family proteins play a critical role in protecting cells from genomic instability and malignant transformation. However, in NB, their activities are often inhibited by interacting proteins such as MDM2. The interplay between p53 family pathway and N-Myc, a key biomarker of poor prognosis, is also a critical factor in NB pathogenesis. Herein, we disclose 1-(dibromomethyl)-3,4,6-trimethoxy-9H-xanthen-9-one (LEM3) as a new p53 family-activating agent with potent NB anticancer activity. At 0.13-2.1 mu M, LEM3 inhibited the growth of several NB cell lines. Its activity was further evidenced in spheroids, patient-derived NB cells, and in a vasculature stiffness-based model of MYCN-amplified NB cells. This growth-inhibitory effect was associated with cell cycle arrest and apoptosis, in SH-SY5Y and SK-N-BE(2) NB cells, without apparent acquisition of resistance. LEM3 inhibited cell migration and invasion and reduced the expression of NB-related prognostic markers, particularly MYCN mRNA and protein levels. LEM3 released p53, TAp63, and TAp73 from their interaction with MDM2 both in a yeast-based assay and NB cells; for p53, this led to increased protein stabilization, DNA-binding ability, and transcriptional activity. Fluorescence quenching and docking analyses suggested that LEM3 binds to p53, TAp63, and TAp73 at the MDM2-binding site within their transactivation domain. LEM3 also synergies with doxorubicin and cisplatin in NB cells. Given the central role of the p53 family MDM2-MYCN axis in NB pathogenesis, our findings support LEM3 as a promising compound for advancing NB targeted therapy.

This review delves into the significant cellular and molecular responses triggered by UVR exposure in human skin, emphasizing the pivotal role of mutant p53 (mutp53) in the carcinogenic process elicited by radiation. By underlining the role of a functional p53 in safeguarding skin cells from UVR-induced damage, this work underscores the potential significance of targeting mutp53, aiming to restore its wild-type-like activity (reactivation), as a protective strategy against skin cancer (SC), particularly NMSC. Most importantly, an interesting crosstalk between p53 and its vitamin D receptor (VDR) transcriptional target is also highlighted in the suppression of skin carcinogenesis, which opens the way to promising chemopreventive strategies involving synergistic combinations between mutp53 reactivators and vitamin D. Collectively, this review not only opens new avenues for future research, but also offers promising prospects for the development of novel beneficial approaches in the field of SC.

Background Doxorubicin is an important anticancer drug, however, elicits dose-dependently cardiomyopathy. Given its mode of action, i.e. topoisomerase inhibition and DNA damage, we investigated genetic events associated with cardiomyopathy and searched for mechanism-based possibilities to alleviate cardiotoxicity. We treated rats at clinically relevant doses of doxorubicin. Histopathology and transmission electron microscopy (TEM) defined cardiac lesions, and transcriptomics unveiled cardiomyopathy-associated gene regulations. Genomic-footprints revealed critical components of Abl1-p53-signaling, and EMSA-assays evidenced Abl1 DNA-binding activity. Gene reporter assays confirmed Abl1 activity on p53-targets while immunohistochemistry/immunofluorescence microscopy demonstrated Abl1, p53&p73 signaling. Results Doxorubicin treatment caused dose-dependently toxic cardiomyopathy, and TEM evidenced damaged mitochondria and myofibrillar disarray. Surviving cardiomyocytes repressed Parkin-1 and Bnip3-mediated mitophagy, stimulated dynamin-1-like dependent mitochondrial fission and induced anti-apoptotic Bag1 signaling. Thus, we observed induced mitochondrial biogenesis. Transcriptomics discovered heterogeneity in cellular responses with minimal overlap between treatments, and the data are highly suggestive for distinct cardiomyocyte (sub)populations which differed in their resilience and reparative capacity. Genome-wide footprints revealed Abl1 and p53 enriched binding sites in doxorubicin-regulated genes, and we confirmed Abl1 DNA-binding activity in EMSA-assays. Extraordinarily, Abl1 signaling differed in the heart with highly significant regulations of Abl1, p53 and p73 in atrial cardiomyocytes. Conversely, in ventricular cardiomyocytes, Abl1 solely-modulated p53-signaling that was BAX transcription-independent. Gene reporter assays established Abl1 cofactor activity for the p53-reporter PG13-luc, and ectopic Abl1 expression stimulated p53-mediated apoptosis. Conclusions The tyrosine kinase Abl1 is of critical importance in doxorubicin induced cardiomyopathy, and we propose its inhibition as means to diminish risk of cardiotoxicity.

The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations.

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Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins.

The long non-coding RNA EPR is expressed in epithelial tissues, binds to chromatin and controls distinct biological activities in mouse mammary gland cells. Because of its high expression in the intestine, in this study we have generated a colon-specific conditional targeted deletion (EPR cKO) to evaluate EPR in vivo functions in mice. EPR cKO mice display epithelium hyperproliferation, impaired mucus production and secretion, as well as inflammatory infiltration in the proximal portion of the large intestine. RNA sequencing analysis reveals a rearrangement of the colon crypt transcriptome with strong reduction of goblet cell-specific factors including those involved in the synthesis, assembly, transport and control of mucus proteins. Further, colon mucosa integrity and permeability are impaired in EPR cKO mice, and this results in higher susceptibility to dextran sodium sulfate (DSS)-induced colitis and tumor formation. Human EPR is down-regulated in human cancer cell lines as well as in human cancers, and overexpression of EPR in a colon cancer cell line results in enhanced expression of pro-apoptotic genes. Mechanistically, we show that EPR directly interacts with select genes involved in mucus metabolism whose expression is reduced in EPR cKO mice and that EPR deletion causes tridimensional chromatin organization changes.

Clinical characteristics of the tumor specimens form which primary cancer stem cells were isolated.

HuR expression ablation does not induce anoikis.

Primers used in PCR reactions. Gene silencing and over-expression tools.

Cell migration/adhesion assays on HuR KD cells and quality controls on the their xenotransplants.

Rescue of phenotype by CD133 silencing in HuR KD cells and assessment of HuR binding to CD133 mRNA.